US10265355B2 - Shigatoxin-producing F18 type E. coli bacteriophage esc-COP-1 and use thereof for inhibiting proliferation of shigatoxin-producing F18 type E. coli - Google Patents
Shigatoxin-producing F18 type E. coli bacteriophage esc-COP-1 and use thereof for inhibiting proliferation of shigatoxin-producing F18 type E. coli Download PDFInfo
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- US10265355B2 US10265355B2 US15/538,573 US201515538573A US10265355B2 US 10265355 B2 US10265355 B2 US 10265355B2 US 201515538573 A US201515538573 A US 201515538573A US 10265355 B2 US10265355 B2 US 10265355B2
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/02—Non-contaminated water, e.g. for industrial water supply
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/04—Disinfection
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2305/00—Use of specific compounds during water treatment
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
- C12N2795/10121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
- C12N2795/10132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
Definitions
- the present invention relates to a bacteriophage isolated from the nature that infects and kills Shigatoxin-producing type F18 E. coli , and a method for preventing and treating the infections of Shigatoxin-producing type F18 E. coli using a composition comprising the bacteriophage as an active ingredient. More particularly, the present invention relates to a Myoviridae bacteriophage Esc-COP-1 that is isolated from the nature and can kill specifically Shigatoxin-producing type F18 E. coli strains, which has a genome represented by the nucleotide sequence of SEQ. ID. NO: 1 (Accession NO: KCTC 12662BP), and a method for preventing the infections of Shigatoxin-producing type F18 E. coli and thereafter treating them using the composition comprising said bacteriophage as an active ingredient.
- E. coli Escherichia coli
- non-pathogenic E. coli pathogenic E. coli
- pathogenic E. coli attaches on intestinal wall through pili to proliferate and produces enterotoxins causing diarrhea.
- the pathogenic E. coli affects various kinds of livestock regardless of ages and gives rise to diarrhea, a notable symptom, possibly leading to high mortality due to dehydration. In Korea, this diarrhea is reported to occur in almost livestock farms. Moreover, in case of mixed infections by Rotavirus, Coronavirus, protozoa Coccidium and the like, this outbreak is stimulated because of damaging enteral mucosa and symptoms is highly aggravated, compared to the case of single infections. There are several pathogenic E. coli strains causing diarrhea. Above all, Shigatoxin-producing type F18 Escherichia coli is often reported to provoke severe diarrhea and edema in pigs. The Shigatoxin-producing type F18 E.
- coli attaches on intestinal epithelial cells through pili (F18) so as to secrete Shigatoxin.
- the secreted toxin is absorbed into blood vessels to increase blood pressure and injure arterioles, thereby generating edema in each part of a body and accompanying convulsion, paralysis and the like.
- Considering a significant damage in livestock industry by the Shigatoxin-producing type F18 E. coli it is urgently requested to develop a method for preventing and treating such infections effectively.
- a variety of antibiotics have been used to prevent or treat such infections of Shigatoxin-producing type F18 E. coli .
- an efficient alternative is urgently requested.
- Bacteriophages are an extremely small microorganism that infects bacteria, which are called phage in short. Once bacteriophage infects bacteria, the bacteriophage is proliferated in the inside of the bacterial cell. After full proliferation, the progenies destroy the bacterial cell wall to escape from the host, suggesting that the bacteriophage has bacteria killing ability.
- the bacteriophage infection is characterized by high specificity, so that a certain bacteriophage infects only a specific bacterium. That is, the bacterium that can be infected by certain bacteriophage is limited, suggesting that bacteriophage can kill only a specific bacterium and cannot harm other bacteria.
- a French bacteriologist d'Herelle found out that Shigella disentriae in the filtrate of dysentery patient feces melted by something, and further studied about this phenomenon.
- bacteriophages Owing to the unique capability of bacteriophage to kill bacteria, bacteriophages have been studied and anticipated as a better method to treat bacterial infections. However, after penicillin was found by Fleming, studies on bacteriophages had been only continued in some of Eastern European countries and the former Soviet Union because of the universalization of antibiotics. After the year of 2000, the merit of the conventional antibiotics faded because of the increase of antibiotic-resistant bacteria. So, bacteriophages are once again spotlighted as a new anti-bacterial agent that can replace the conventional antibiotics.
- the present inventors tried to develop a composition applicable for the prevention or treatment of Shigatoxin-producing type F18 E. coli infections by using a bacteriophage that is isolated from the nature and can kill Shigatoxin-producing type F18 E. coli selectively, and further to establish a method for preventing or treating the infections of Shigatoxin-producing type F18 E. coli using the composition.
- the present inventors isolated bacteriophages suitable for this purpose and secured the nucleotide sequence of the genome that distinguishes the bacteriophage of the present invention from other bacteriophages. Then, we have developed a composition comprising the isolated bacteriophage as an active ingredient, and confirmed that this composition could be efficiently used for the prevention and treatment of Shigatoxin-producing type F18 E. coli infections, leading to the completion of the present invention.
- the present invention provides a Myoviridae bacteriophage ESC-COP-1 that is isolated from the nature and can kill specifically Shigatoxin-producing type F18 E. coli , which has the genome represented by the nucleotide sequence of SEQ. ID. NO: 1 (Accession NO: KCTC 12662BP), and a method for preventing and treating the infections of Shigatoxin-producing type F18 E. coli using a composition comprising the bacteriophage as an active ingredient.
- the bacteriophage Esc-COP-1 has been isolated by the present inventors and then deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology in Aug. 21, 2014 (Accession NO: KCTC 12662BP).
- the present invention also provides a disinfectant, a drinking water additive, and a feed additive applicable for the prevention or treatment of Shigatoxin-producing type F18 E. coli infections, which comprises the bacteriophage Esc-COP-1 as an active ingredient.
- the composition of the present invention can be utilized for the prevention and treatment of E. coli diarrhea caused by Shigatoxin-producing type F18 E. coli .
- the E. coli diarrhea includes symptoms caused by the E. coli infections accompanying fever, diarrhea and the like.
- treatment indicates (i) to suppress the diarrhea caused by Shigatoxin-producing type F18 E. coli ; and (ii) to relieve the diarrhea caused by Shigatoxin-producing type F18 E. coli.
- isolation indicates all the actions to separate the bacteriophage by using diverse experimental techniques and to secure the characteristics that can distinguish this bacteriophage from others, and further includes the action of proliferating the bacteriophage via bioengineering techniques so as to make it useful.
- the pharmaceutically acceptable carrier included in the composition of the present invention is the one that is generally used for the preparation of a pharmaceutical formulation, which is exemplified by lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silcate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but not always limited thereto.
- the composition of the present invention can additionally include lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, and preservatives, in addition to the above ingredients.
- the bacteriophage Esc-COP-1 is included as an active ingredient.
- the bacteriophage Esc-COP-1 is included at the concentration of 1 ⁇ 10 1 pfu/ml ⁇ 1 ⁇ 10 30 pfu/ml or 1 ⁇ 10 1 pfu/g ⁇ 1 ⁇ 10 30 pfu/g, and preferably at the concentration of 1 ⁇ 10 4 pfu/ml ⁇ 1 ⁇ 10 15 pfu/ml or 1 ⁇ 10 4 pfu/g ⁇ 1 ⁇ 10 15 pfu/g.
- composition of the present invention can be formulated by the method that can be performed by those in the art by using a pharmaceutically acceptable carrier and/or excipient in the form of unit dose or in a multi-dose container.
- the formulation can be in the form of solution, suspension or emulsion in oil or water-soluble medium, extract, powder, granule, tablet or capsule.
- a dispersing agent or a stabilizer can be additionally included.
- composition of the present invention can be prepared as a disinfectant, a drinking water additive, or a feed additive according to the purpose of use, but not always limited thereto.
- the method for preventing and treating the infections of Shigatoxin-producing type F18 E. coli using this composition comprising the bacteriophage Esc-COP-1 as an active ingredient, have the advantage of high specificity to Shigatoxin-producing type F18 E. coli , compared with the conventional methods based on the chemical materials including the conventional antibiotics. That means, the composition of the present invention can be used for preventing or treating the infections of Shigatoxin-producing type F18 E. coli specifically without affecting other useful residential bacteria, and accordingly has fewer side effects. In general, when chemical materials such as antibiotics are used, the general residential bacteria are also damaged to weaken immunity in animals with carrying various side effects. In the meantime, the composition of the present invention uses the bacteriophage isolated from the nature as an active ingredient, so that it is very nature-friendly.
- FIG. 1 is an electron micrograph showing the morphology of the bacteriophage Esc-COP-1.
- FIG. 2 is a photograph illustrating the capability of the bacteriophage Esc-COP-1 to kill Shigatoxin-producing type F18 E. coli .
- the clear zone on the dish is the formation of plaque by lysis of bacteria cells.
- Example 1 Isolation of Bacteriophage Capable of Killing Shigatoxin-Producing Type F18 E. coli
- the isolation procedure of the bacteriophage is described in detail hereinafter.
- the collected sample was added to the TSB (Tryptic Soy Broth) medium (pancreatic digest of casein, 17 g/L; papaic digest of soybean, 3 g/L; dextrose, 2.5 g/L; sodium chloride, 5 g/L; dipotassium phosphate, 2.5 g/L) inoculated with Shigatoxin-producing type F18 E. coli at the ratio of 1/1000, followed by shaking culture at 37° C. for 3 ⁇ 4 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes and supernatant was recovered.
- TSB Tryptic Soy Broth
- the recovered supernatant was inoculated with Shigatoxin-producing type F18 E. coli at the ratio of 1/1000, followed by shaking culture at 37° C. for 3 ⁇ 4 hours.
- the above procedure was repeated total 5 times in order to increase the titer of the bacteriophage.
- the culture solution proceeded to centrifugation at 8,000 rpm for 20 minutes and the resulting supernatant was recovered.
- the recovered supernatant was filtrated by using a 0.45 ⁇ m filter. The obtained filtrate was used in spot assay for examining whether or not the bacteriophage capable of killing Shigatoxin-producing type F18 E. coli was included therein.
- TSB medium was inoculated with Shigatoxin-producing type F18 E. coli at the ratio of 1/1000, followed by shaking culture at 37° C. for overnight.
- 3 ml (1.5 of OD 600 ) of the culture broth of Shigatoxin-producing type F18 E. coli prepared above was spread on the TSA (Tryptic Soy Agar; pancreatic digest of casein, 17 g/L; papaic digest of soybean, 3 g/L; sodium chloride, 5 g/L; agar, 15 g/L) plate. The plate stood in a chamber for about 30 minutes to dry.
- the bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing Shigatoxin-producing type F18 E. coli .
- the conventional plaque assay was used for the isolation of pure bacteriophages. In detail, a plaque formed in the course of the plaque assay was picked up by using a sterilized tip, which was then added to the culture solution of Shigatoxin-producing type F18 E. coli , followed by culturing for 4 ⁇ 5 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. The recovered supernatant was inoculated with Shigatoxin-producing type F18 E.
- the above procedure was repeated at least 5 times. Then, centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. Plaque assay was performed with the obtained supernatant.
- the pure bacteriophage isolation is not completed by one-time procedure, so the above procedure was repeated by using the plague formed above. After at least 5 times of repeated procedure, the solution containing the pure bacteriophage was obtained. The procedure for the isolation of the pure bacteriophage was generally repeated until the generated plaques became similar in sizes and morphologies.
- the observation under electron microscope was performed by the conventional method. Briefly, the solution containing the pure bacteriophage was loaded on copper grid, followed by negative staining with 2% uranyl acetate. After drying thereof, the morphology was observed under transmission electron microscope.
- the electron micrograph of the bacteriophage isolated in the present invention is presented in FIG. 1 . From the morphological observation, the bacteriophage isolated above was identified as belonging to the family Myoviridae.
- the solution containing the pure bacteriophage confirmed above proceeded to purification.
- the culture broth of Shigatoxin-producing type F18 E. coli was added to the solution containing the pure bacteriophage at the volume of 1/50 of the total volume of the bacteriophage solution, followed by culturing again for 4 ⁇ 5 hours.
- centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. This procedure was repeated 5 times to obtain a solution containing enough numbers of the bacteriophage.
- the supernatant obtained from the final centrifugation was filtered by a 0.45 ⁇ m filter, followed by the conventional polyethylene glycol (PEG) precipitation.
- PEG polyethylene glycol
- PEG and NaCl were added to 100 ml of the filtrate until reaching 10% PEG 8000/0.5 M NaCl, which stood at 4° C. for 2 ⁇ 3 hours. Then, centrifugation was performed at 8,000 rpm for 30 minutes to obtain the bacteriophage precipitate. The resulting bacteriophage precipitate was resuspended in 5 ml of buffer (10 mM Tris-HCl, 10 mM MgSO 4 , 0.1% Gelatin, pH 8.0). This solution was called as the bacteriophage suspension or bacteriophage solution.
- the pure bacteriophage purified above was collected, which was named as the bacteriophage Esc-COP-1 and then deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology in Aug. 21, 2014 (Accession NO: KCTC 12662BP).
- the genome of the bacteriophage Esc-COP-1 was separated as follows.
- the genome was separated from the bacteriophage suspension obtained in Example 1.
- DNase I and RNase A were added 200 U each to 10 ml of the bacteriophage suspension, which was incubated at 37° C. for 30 minutes. 30 minutes later, to remove the DNase I and RNase A activity, 500 ⁇ l of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added thereto, which was incubated for 10 minutes. The suspension was further incubated at 65° C.
- EDTA ethylenediaminetetraacetic acid
- the upper layer was obtained, to which isopropyl alcohol was added at the volume of 1.5 times the volume of the upper layer, followed by centrifugation at 13,000 rpm for 10 minutes to precipitate the genome of the bacteriophage. After collecting the precipitate, 70% ethanol was added to the precipitate, followed by centrifugation at 13,000 rpm for 10 minutes to wash the precipitate. The washed precipitate was recovered, vacuum-dried and then dissolved in 100 ⁇ l of water. This procedure was repeated to obtain a sufficient amount of the bacteriophage Esc-COP-1 genome.
- the nucleotide sequence of the genome of the bacteriophage Esc-COP-1 obtained above was analyzed by Next Generation Sequencing (NGS) using illumina Mi-Seq device at National Instrumentation Center for Environmental Management, Seoul National University. As a result, it is suggested that the final genome of bacteriophage Esc-COP-1 has 169,727 bp of size and the nucleotide sequence of the whole genome has SEQ. ID. NO: 1.
- E. coli bacteriophage RB51 Genbank Accession NO: FJ839693.1
- E. coli bacteriophage RB68 Genbank Accession NO: KM607004. 1
- their genome sizes were discriminated one another.
- the whole genome of bacteriophage Esc-COP-1 was determined to have 169,727 bp of size, while whole genome of E. coli bacteriophage vB_EcoM_ACG-C40 had 167,396 bp of size, that of E. coli bacteriophage RB14 had 165,429 bp of size, that of E.
- coli bacteriophage HY01 had 166,977 bp of size, that of E. coli bacteriophage RB51 had 168,394 bp of size and that of E. coli bacteriophage RB68 had 168,401 bp of size distinctly. Furthermore, the number of ORFs (Open Reading Frame) within the genome of bacteriophage Esc-COP-1 was determined to 275 ORFs, while the number of ORFs within E. coli bacteriophage vB_EcoM_ACG-C40 was 273 ORFs, that of E. coli bacteriophage RB14 was 274ORFs, that of E.
- ORFs Open Reading Frame
- E. coli bacteriophage HY01 was 257 ORFs, and that of E. coli bacteriophage RB68 was 276 ORFs distinctly. But the number of ORFs within the genome of E. coli bacteriophage RB51 was 275 ORFs, which was same with that of bacteriophage Esc-COP-1. Nevertheless, the ORFs arrangement within the genome of E. coli bacteriophage RB51 was very different from that of bacteriophage Esc-COP-1.
- the killing ability of the isolated bacteriophage Esc-COP-1 against Shigatoxin-producing type F18 E. coli was investigated. To do so, the formation of clear zone was observed by the spot assay by the same manner as described in Example 1.
- the Shigatoxin-producing type F18 E. coli used for this investigation were total 10 strains which had been isolated and identified as Shigatoxin-producing type F18 E. coli previously by the present inventors.
- the bacteriophage Esc-COP-1 demonstrated the killing ability against 9 strains of the Shigatoxin-producing type F18 E. coli used in this experiment.
- the representative result of the killing ability test is shown in FIG. 2 .
- the activity of the bacteriophage Esc-COP-1 to kill Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus plantarum, Streptococcus uberis and Pseudomonas aeruginosa was also investigated. As a result, it is decided that the bacteriophage Esc-COP-1 did not have the killing activity against these microorganisms.
- the bacteriophage Esc-COP-1 has the specific ability to kill Shigatoxin-producing type F18 E. coli and a broad antibacterial spectrum against Shigatoxin-producing type F18 E. coli , suggesting that the bacteriophage Esc-COP-1 of the present invention could be used as an active ingredient of the composition for preventing and treating the infections of Shigatoxin-producing type F18 E. coli.
- the bacteriophage Esc-COP-1 not only inhibited the growth of Shigatoxin-producing type F18 E. coli but also could kill them. Therefore, the bacteriophage Esc-COP-1 can be used as an active ingredient of the composition for preventing the infections of Shigatoxin-producing type F18 E. coli.
- the diarrhea index was set as follows according to Fecal Consistency (FC) score (normal: 0, loose stool: 1, moderate diarrhea: 2, and severe diarrhea: 3). The results are shown in Table 2.
- the bacteriophage Esc-COP-1 of the present invention could be very effective to treat the infections of Shigatoxin-producing type F18 E. coli.
- Feed additive containing bacteriophage Esc-COP-1 at a concentration of 1 ⁇ 10 8 pfu/g was prepared using the bacteriophage Esc-COP-1 solution.
- the preparation method thereof was as follows: Maltodextrin (40%, w/v) was added to the bacteriophage solution and then, trehalose was added to reach 10% of final concentration. After mixing well, the mixture was freeze-dried. Lastly, the dried mixture was grinded into fine powders. The drying process above can be replaced with vacuum-drying, drying at warm temperature, or drying at room temperature. To prepare the control feed additive for comparison, feed additive that did not contain the bacteriophage but contained buffer (10 mM Tris-HCl, 10 mM MgSO 4 , 0.1% Gelatin, pH 8.0) only was prepared.
- Drinking water additive and disinfectant are different in intended use but same in the composition, so they have been prepared by the same manner.
- Drinking water additive (or disinfectant) containing bacteriophage Esc-COP-1 at a concentration of 1 ⁇ 10 8 pfu/ml was prepared using the bacteriophage Esc-COP-1 solution.
- the bacteriophage ESC-COP-1 solution was added to buffer solution to reach 1 ⁇ 10 8 pfu/ml, which was mixed well.
- the above buffer solution itself was used as the drinking water additive (or disinfectant) that did not contain the bacteriophage.
- the prepared two kinds of drinking water additives were diluted in water at the ratio of 1:1000, and then used as drinking water or disinfectant.
- Example 6 and Example 7 The effect of the feeds, drinking water, and disinfectant prepared in Example 6 and Example 7 on pig farming was investigated. Particularly, the investigation was focused on diarrhea conditions by fecal consistency score used in Example 5.
- Total 30 piglets were grouped into three groups, and each group was composed of 10 piglets (group A: feed test group, group B: drinking water test group; and group C: disinfectant test group). The experiment was continued for 2 weeks. Each group was divided by two sub-groups comprising 5 piglets each.
- the sub-groups were divided according to the treatment of the bacteriophage Esc-COP-1 or not (sub-group- ⁇ circle around (1) ⁇ : treated with the bacteriophage Esc-COP-1; and sub-group- ⁇ circle around (2) ⁇ : not-treated with the bacteriophage).
- the piglets used in this experiment were weaning pigs at 20 days of age and raised in a separated room placed at a sufficient distance from each other. Each sub-group was divided and named as shown in Table 3.
- Feeds were provided according to the conventional feed supply method as presented in Table 3 with the feeds prepared in Example 6.
- Drinking water was provided according to the conventional water supply method as presented in Table 3 with the drinking water prepared in Example 7.
- Disinfectant was treated three times a week with taking turns with the conventional disinfectant. That is, on the day when the disinfectant of the present invention was sprayed, the conventional disinfectant was not treated. The results are shown in Table 4.
Abstract
Description
TABLE 1 |
Inhibition of growth of Shigatoxin-producing type F18 |
E. coli |
OD600 |
Culturing | Culturing | Culturing | |||
item | 0 min. | 60 min. | 120 min. | ||
(−) bacteriophage | 0.5 | 1.5 | 2.1 | ||
solution | |||||
(+) bacteriophage | 0.5 | 0.4 | 0.3 | ||
solution | |||||
TABLE 2 |
Fecal Consistency score |
Days after Shigatoxin-producing | |
type F18 E. coli challenge |
0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
Control group | 2.25 | 2.5 | 2.5 | 2.25 | 2 | 2 | 1.5 | 1.5 |
(− bacteriophage | ||||||||
solution) | ||||||||
Experimental group | 2.5 | 1.75 | 1 | 0.5 | 0.25 | 0.25 | 0 | 0 |
(+ bacteriophage | ||||||||
solution) | ||||||||
TABLE 3 |
Sub-groups of pig farming experiment |
Sub-group |
Treated with the | ||||
bacteriophage Esc- | Not-treated with | |||
Item | COP-1 | the bacteriophage | ||
Fed with feeds | A-{circle around (1)} | A-{circle around (2)} | ||
Provided with | B-{circle around (1)} | B-{circle around (2)} | ||
drinking water | ||||
Treated with | C-{circle around (1)} | C-{circle around (2)} | ||
disinfectant | ||||
TABLE 4 |
Fecal consistency score of pig farming experiment |
Group | Fecal consistency score |
d1 | d2 | d3 | d4 | d5 | d6 | d7 | d8 | d9 | d10 | d11 | d12 | d13 | d14 | |
A-{circle around (1)} | 0 | 0 | 0.2 | 0.2 | 0 | 0 | 0 | 0.2 | 0.2 | 0 | 0 | 0 | 0.2 | 0 |
A-{circle around (2)} | 0 | 0.2 | 0.4 | 0.4 | 0.2 | 0.2 | 0.2 | 0.4 | 0.4 | 0.4 | 0.4 | 0.2 | 0.2 | 0.2 |
B-{circle around (1)} | 0.2 | 0 | 0 | 0 | 0 | 0 | 0.2 | 0 | 0 | 0 | 0 | 0.2 | 0 | 0 |
B-{circle around (2)} | 0.2 | 0.2 | 0.2 | 0.4 | 0.4 | 0.4 | 0.2 | 0.2 | 0.4 | 0.4 | 0.2 | 0.4 | 0.4 | 0.2 |
C-{circle around (1)} | 0.2 | 0.2 | 0 | 0 | 0 | 0.2 | 0.2 | 0.2 | 0 | 0 | 0.2 | 0 | 0 | 0 |
C-{circle around (2)} | 0 | 0.2 | 0.2 | 0.2 | 0.4 | 0.4 | 0.2 | 0.4 | 0.4 | 0.2 | 0.2 | 0.2 | 0.2 | 0.4 |
Claims (2)
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Application Number | Priority Date | Filing Date | Title |
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KR1020140192983A KR101649851B1 (en) | 2014-12-30 | 2014-12-30 | Novel Shigatoxin-producing Escherichia coli type F18 bacteriophage Esc-COP-1 and its use for preventing proliferation of Shigatoxin-producing Escherichia coli type F18 |
KR10-2014-0192983 | 2014-12-30 | ||
PCT/KR2015/014331 WO2016108541A1 (en) | 2014-12-30 | 2015-12-28 | Novel shigatoxin-producing f18 type e. coli bacteriophage esc-cop-1 and use thereof for inhibiting proliferation of shigatoxin-producing f18 type e. coli |
Publications (2)
Publication Number | Publication Date |
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US20170340686A1 US20170340686A1 (en) | 2017-11-30 |
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CO2017006495A2 (en) | 2017-09-20 |
KR101649851B1 (en) | 2016-08-30 |
CN107208068B (en) | 2021-04-20 |
US20170340686A1 (en) | 2017-11-30 |
RU2662985C1 (en) | 2018-07-31 |
WO2016108541A1 (en) | 2016-07-07 |
CN107208068A (en) | 2017-09-26 |
MX2017008563A (en) | 2017-10-26 |
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BR112017013238A2 (en) | 2018-08-07 |
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